Transgenerational effects of environmental toxins, such as endocrine disrupters, significantly amplify the impact and health hazards of these compounds. The transgenerational nature of the actions of these compounds suggests an epigenetic effect on the germ-line. The current proposal is designed to investigate this transgenerational epigenetic phenomenon on male reproduction. Endocrine disrupters have been shown to influence male reproduction by causing abnormal sperm numbers and fertility. One of the most sensitive periods to endocrine disrupter exposure is during embryonic development. The objective of the proposed research is to investigate the mechanism of action of model endocrine disrupters on male reproduction with a focus on testis development. A rodent model (i.e. rat) system is used to provide insight into the mechanistic aspects of endocrine disrupter action. The model endocrine disruptors tested are methoxychlor that has metabolites that are both weak estrogenic and anti-androgenic compounds, and vinclozolin, which is an anti-androgenic compound. Therefore, these model endocrine disruptors allow consideration of both estrogenic and anti-androgenic endocrine disrupter actions. The objective is to obtain insight into the molecular, cellular and physiological (i.e. systems biology) actions of endocrine disruptors on male reproduction. The hypothesis tested is that transient embryonic in utero exposure to an endocrine disruptor influences the embryonic testis transcriptome and through epigenetic effects (e.g. DNA methylation) results in abnormal germ cell differentiation that subsequently influences adult spermatogenic capacity and male fertility and that this phenotype is transgenerational through the germ-line. Previous studies have shown that methoxychlor and vinclozolin can effect embryonic testis development at the time of testis morphogenesis and that this causes an increase in germ cell apoptosis in the adult. Interestingly, observations suggest this abnormal spermatogenesis is transgenerational and may be due to altered DNA methylation of the germ-line through an epigenetic action of the endocrine disrupter. Preliminary studies have also demonstrated that altered gene expression of paracrine growth factors directly influence testis development at the time of endocrine disrupter action. Abnormal testis development and germ cell differentiation caused by endocrine disruptors may in part be due to inappropriate control of the testis transcriptome and abnormal germ cell development. The experimental approach to test the above hypothesis consists of the following specific aims: 1) Investigate the transgenerational effects of endocrine disruptors on testis development and gametogenesis. 2) Determine the mechanism of endocrine disrupter actions through analysis of epigenetic effects (e.g. DNA methylation) on the testis and germ-line. 3) Determine the actions of transient embryonic exposure to endocrine disruptors on the embryonic and postnatal testis transcriptome. Information obtained from the proposed research will determine how environmental toxins (i.e. vinclozolin and methoxychlor) may impair male fertility by adversely affecting the embryonic testis transcriptome and germ-line DNA methylation. The completion of these studies will provide insight into the mechanistic aspects of how embryonic exposure to endocrine disruptors cause a transgenerational effect on adult male reproductive function. The proposed research will be used to extrapolate and provide insight into the impact of endocrine disruptors on human development, reproduction and health. The novel observations of transgenerational epigenetic endocrine disrupter actions on male reproduction critically impacts the potential hazards of these compounds as environmental toxins. The proposed research will thoroughly establish this phenomenon and elucidate the molecular mechanisms involved.